Normal human cells in vitro exhibit a stringent limitation of division capacity. In contrast, tumor-derived and virus-, carcinogen- or irradiation-transformed cells can divide indefinitely (immortal). We do not yet understand the mechanisms that limit the division potential of normal human cells or the changes occurring to yield immortal cells. However, from cell hybrid studies we have found that the immortal phenotype results from recessive changes in normal cell growth control. We have exploited this fact to separate twenty four different immortal cell lines into four complementation groups for indefinite division. This indicates that there are at least four paths to immortality and that at least four genes or sets of genes are involved in normal cell growth control. Using the technique of microcell mediated chromosome transfer, we have determined that the introduction of a normal human chromosome 4 into three immortal human cell lines that assign to complementation group B, results in loss of proliferation. This is a specific effect to the group B cell lines, because the division capability of immortal cell lines assigned to the other groups is not affected by this chromosome, and a normal chromosome 11 has no effect on any of the immortal cell lines tested. These results indicate that there is a cell senescence related gene(s) on human chromosome 4. We have also found that a normal chromosome 1 induces loss of proliferation in one immortal human cell line that assigns to group C., In this proposal, we plan to take multipronged approaches to clone the gene on chromosome 4 and study regulation of its expression during senescence and immortalization. We will also determine whether chromosome 1 affects the proliferation potential of other immortal human cell lines that assign to group C. If it does, this would indicate that another cellular senescence gene(s) is present on this chromosome and we will attempt to localize the region on chromosome 1 that is involved.